Template-dependent nucleic acid synthesis methods using the Polymerase Chain Reaction (PCR) method have served as a major driving force for research in bioscience fields in recent years. The PCR method has made it possible to exponentially amplify nucleic acids composed of a nucleotide sequence complementary to a template by using a small amount of double-stranded nucleic acid as template. PCR is currently widely used as a tool for gene cloning and detection. In the PCR method, one set of primers comprising nucleotide sequences complementary to both ends of a target nucleotide sequence is used. One of the primers is designed to anneal to the elongation product generated by the other primer. In this manner, a synthesis reaction progresses in which annealing to a mutual elongation product and complementary strand synthesis are repeated, enabling exponential amplification to be achieved.
In the PCR method, complex temperature control is essential. A special reaction apparatus must be used to accommodate this complex temperature control. Thus, it is difficult to perform PCR at hospital bedsides or outdoors. In addition, improvement of reaction specificity has been an important problem for known complementary strand synthesis reactions. For example, in the PCR method, when a complementary strand synthesis product is used as a new template, the region to which the primer anneals is not a nucleotide sequence derived from the sample in the strict sense, but rather is merely a copy of the nucleotide sequence of the primer. Thus, it is typically difficult to recognize slight differences in nucleotide sequences using a PCR primer.
As one method for solving these problems, the LAMP method was invented (Loop-mediated isothermal amplification) (Nucleic Acid Res. 2000, Vol. 28, No. 12 e63, WO 00/28082). The LAMP method makes it possible to anneal to a template polynucleotide its own 3′-end to serve as a starting point for complementary strand synthesis, while also enabling an isothermal complementary strand synthesis reaction by combining a primer that is annealed to the loop formed at this time. In addition, in the LAMP method, since the 3′-end anneals to a region derived from the sample, a nucleotide sequence checking mechanism functions repeatedly. As a result, it has become possible to identify even slight differences in nucleotide sequences.
When detecting a target nucleotide sequence based on a complementary strand synthesis reaction such as LAMP or PCR, there is a close relationship between the time required for the reaction and detection sensitivity. In other words, allowing the reaction to proceed for as long a time as possible until the reaction reaches a plateau is a condition for achieving high detection sensitivity. In the case of known complementary strand synthesis reactions like LAMP and PCR, the reaction reaches a plateau in about 1 hour. In other words, it may be said that a reaction time of about 1 hour is required in order to obtain maximum sensitivity. Although a reaction time of 1 hour is not that long, it would be even more useful if an even shorter reaction time can be realized without sacrificing detection sensitivity or procedural ease.
Following the identification of the genome draft, science is entering a post-genome era. There is a growing need for analyzing single nucleotide polymorphisms (SNPs) and gene function as well as gene diagnosis based on the results of those analyses. Thus, the development of a technology that enables gene nucleotide sequences to be analyzed more accurately and rapidly is becoming an important issue not only in terms of rapidly carrying out functional analysis, but also with respect to practical application of the results of gene function analysis in actual clinical settings.